Epidemiological characteristics, virulence potential, antimicrobial resistance profiles, and phylogenetic analysis of Aeromonas caviae isolated from extra-intestinal infections.

Objective: Aeromonas caviae (A. caviae) is one of the major etiological agents in human intestinal infections reported to be associated with a broad spectrum of extra-intestinal infections with increasing incidence over recent years. Although previous studies have established its significance as a causative agent of both bloodstream and gastrointestinal infections, the characteristics of A. caviae that cause extra-intestinal infections remain unilluminated.In this single-center retrospective study, we investigated epidemiological characteristics, antimicrobial resistance genes and phenotypes, virulence genes, and phyloevolution of 47 clinical A. caviae isolated from patients with extra-intestinal infections from 2017 to 2020. Methods: A. caviae strains were identified by biochemical tests and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS), ultimately confirmed to species level by whole-genome sequencing (WGS). Antimicrobial resistance and virulence genes were identified using the Comprehensive Antibiotic Resistance Database (CARD) and the virulence factor database (VFDB), respectively. Phylogenetic analysis of 47 clinical strains was performed by combining with 521 A. caviae strains from NCBI database. Results: A. caviae was an opportunistic pathogen in immunocompromised patients, especially those with underlying hepatobiliary diseases and malignancies. 19 out of 47 isolates were identified as multidrug resistance (MDR) strains. Piperacillin-tazobactam, levofloxacin, gentamicin, amikacin with a resistance rate of less than 10% remained as options to treat extra-intestinal infections. 24 out of 47 isolates exhibited non-susceptibility to cephalosporins and cephamycins, all of which carried ß-lactamase gene, including bla MOX, bla PER-3, bla OXA, bla NDM, and bla CphA. Most stains (98%, 46/47) carried at least one of the virulence genes, but extra-intestinal infections had a low mortality rate. Phylogenetic analysis indicated the risk of nosocomial transmission but revealed no outbreak. However, the emergence of MDR and ß-lactamase resistance genes in extra-intestinal isolates of A. caviae is becoming an increasing risk to public health and requires attention. Conclusions: This study strengthen our understanding of A.caviae isolated from extra-intestinal infections. It may contribute to the management of extra-intestinal infections as well as the prevention and control of drug resistance.

Prevalence and characteristics of surgical site hypervirulent Klebsiella pneumoniae isolates.

BACKGROUND: We aim to determine the prevalence of hypervirulent Klebsiella pneumoniae (hvKp), which causes surgical site infections (SSIs), and describe the microbiological and molecular characteristics of hvKp isolates. METHODS: Non-duplicate K. pneumoniae strains were isolated from wound drainage specimens of postoperative patients at the Chinese PLA General Hospital between September 2008 and July 2017. Antimicrobial susceptibility, string test, pulsed-field gel electrophoresis (PFGE), and genome sequencing analyses were performed. RESULTS: Fifty-one K. pneumoniae strains were isolated from wound drainage specimens collected from postoperative patients. Twenty-six hvKp strains, including 17 (17/37, 46.0%) and 9 (9/14, 64.3%) hvKp strains, were isolated from 37 and 14 patients with SSIs and community-acquired infections (CAIs), respectively. Notably, 4 extended-spectrum beta-lactamase (ESBL)-producing hvKp strains (4/26, 15.4%) and 2 carbapenem-resistant hvKp strains (2/26, 7.7%) were found. Thirteen K1 serotype (13/26, 50.0%) and 7 K2 serotype (7/26, 26.9%) strains were identified. Phylogenetic analysis results showed that 13 K1 serotype isolates exhibited a high degree of clonality, while 7 K2 serotype strains were genetically unrelated. MLST analysis indicated that there was a strong correlation between ST23 and the K1 serotype. ST65, ST86, and ST375 were prevalent in K2 serotype strains. Almost all hvKp strains (24/26, 92.3%) harbored large virulence plasmids with a high degree of homology to pNTUH-K2044 and sizes ranging from 140 to 220 kbp. CONCLUSIONS: HvKp strains were prevalent in SSIs. Effective surveillance and control measures should be implemented to prevent the dissemination of such organisms, including the ESBL-producing and carbapenem-resistant hvKp strains.

Clinical and microbiological characteristics of Cryptococcus gattii isolated from 7 hospitals in China.

BACKGROUND: Infection, even outbreak, caused by Cryptococcus gattii (C. gattii) has been reported in Canada and the United States, but there were sparsely-reported cases of C. gattii in China. Our interest in occurrence, clinical manifestation, laboratory identification and molecular characterization of Chinese C. gattii strains leads us to this research. RESULTS: Out of 254 clinical isolates, initially identified as Cryptococcus neoformans (C. neoformans), eight strains were re-identified as C. gattii. Multi-locus sequence typing (MLST) showed genotype VGI accounted for the most (6 / 8), the other two strains were genotype VGII (VGIIa and VGIIb respectively) with 3 specific spectra of molecular weight about 4342, 8686, 9611 Da by MALDI-TOF MS. The minimal inhibitory concentrations (MICs) of Fluconazole with Yeast one was 2~4 times higher than that with ATB fungus 3 and MICs of antifungal agents against VGII strains were higher than against VGI strains. Comparative proteome analysis showed that 329 and 180 proteins were highly expressed by C. gattii VGI and VGII respectively. The enrichment of differentially expressed proteins was directed to Golgi complex. CONCLUSIONS: Infection by C. gattii in China occurred sparsely. Genotype VGI was predominant but VGII was more resistant to antifungal agents. There was significant difference in protein expression profile between isolates of VGI and VGII C. gattii.

Universal Probe Library based real-time PCR for rapid detection of bacterial pathogens from positive blood culture bottles.

A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.

Predictive value of procalcitonin for excluding bloodstream infection: results of a retrospective study and utility of a rapid, quantitative test for procalcitonin.

OBJECTIVES: To assess retrospectively the diagnostic value of procalcitonin (PCT) in excluding suspected bloodstream infection, establish cut-off values for PCT levels, and compare PCT with other clinical markers. METHODS: The predictive accuracy of different continuous parameters was estimated by univariate analysis of the area under the receiver operating characteristic curve. Optimized cut-off points for the parameters were selected according to the maximum Youden index values, which in turn were used to define positive and negative predictive values of different parameters in diagnosing bloodstream infection. RESULTS: The PCT level yielded a statistically significant area under the receiver operating characteristic curve of 0.765, with a best cut-off value of 0.80 ng/ml (83% sensitivity; 65% specificity, Youden index, J = 0.48). Positive and negative predictive values at this cut-off value were 38% and 94%, respectively. Mann-Whitney U-test revealed significantly higher values for PCT, C-reactive protein and percentage of neutrophils, but not for white blood cell count, in patients with bloodstream infection. CONCLUSIONS: The serum PCT level can potentially be used as surrogate marker to exclude bacteraemia and to inform critical management decisions regarding antibiotic usage, in patients admitted with suspected bloodstream infection.

Nosocomial bloodstream infection in patients caused by Staphylococcus aureus: drug susceptibility, outcome, and risk factors for hospital mortality.

BACKGROUND: Previous studies have different viewpoints about the clinical impact of methicillin resistance on mortality of hospital-acquired bloodstream infection (BSI) patients with Staphylococcus aureus (S. aureus). The objective of this study was to investigate the mortality of hospital-acquired BSI with S. aureus in a military hospital and analyze the risk factors for the hospital mortality. METHODS: A retrospective cohort study was performed in patients admitted to the biggest military tertiary teaching hospital in China between January 2006 and May 2011. All included patients had clinically significant nosocomial BSI with S. aureus. Multivariate Logistic regression analysis was used to identify the risk factors for hospital mortality of patients with S. aureus BSI. RESULTS: One hundred and eighteen patients of more than one year old were identified as clinically and microbiologically confirmed nosocomial bacteraemia due to S. aureus, and 75 out of 118 patients were infected with methicillin-resistant S. aureus (MRSA). The overall mortality of nosocomial S. aureus BSI was 28.0%. Methicillin resistance in S. aureus bacteremia was associated with significant increase in the length of hospitalization and high proportion of inappropriate empirical antibiotic treatment. After Logistic regression analysis, the severity of clinical manifestations (APACHE II score) (odds ratio (OR) 1.22, 95% confidence interval (CI) 1.12 - 1.34) and inadequacy of empirical antimicrobial therapy (OR 0.25, 95%CI 0.09 - 0.69) remained as risk factors for hospital mortality. CONCLUSIONS: Nosocomial S. aureus BSI was associated with high in-hospital mortality. Methicillin resistance in S. aureus has no significant impact on the outcome of patients with staphylococcal bacteremia. Proper empirical antimicrobial therapy is very important to the prognosis.

[Colloidal gold and dot-ELISA rapid tests for screening influenza A virus].

OBJECTIVE: To evaluate the diagnostic efficiency of colloidal gold and dot ELISA rapid tests in clinical screening of influenza A virus. METHODS: The pharyngeal swabs were collected from 297 outpatients suspected of influenza between June and October, 2009 for detection with colloid gold and dot ELISA rapid test, with real-time PCR as the golden methods. The discrepant results of colloid gold and dot ELISA methods were confirmed by sequencing, and the diagnostic efficiency of the two methods was evaluated. RESULTS: Among the 166 samples with influenza A virus infection as confirmed by real-time PCR and sequencing, the diagnostic sensitivity of dot ELISA and colloid gold methods was 54.82% (91/166) and 4.22% (7/166), respectively. The total concordance rate with PCR was 66.67% (Kappa value of 0.35). Among the 133 samples negative for influenza A virus, the specificity of dot ELISA and colloid gold methods was 81.68% (107/131) and 98.47% (129/131), respectively, with a total concordance rate with PCR of 45.79% (Kappa value 0.02). Of the 99 H1N1 influenza samples confirmed by real-time PCR, the detection rate of dot ELISA was 67.3%, whereas that of colloid gold was 5.1%. Out of the 107 dot ELISA-positive but colloid gold-negative samples, 84 were confirmed to be influenza A virus-positive by real-time PCR and sequencing. One sample negative for dot ELISA but positive for colloid gold test was confirmed to be influenza A virus-negative. The detection rate and diagnostic concordance rate for influenza A virus by dot ELISA were significantly higher than those of colloid gold (P<0.05). CONCLUSION: Dot ELISA is better than colloid gold in influenza A virus detection and shows great prospect in clinical screening.

[Genotyping on 47 Staphylococcus aureus strains associated with bloodstream infection.].

OBJECTIVE: To investigate the molecular epidemiological characteristics of Staphylococcus aureus associated with bloodstream infection in hospital. METHODS: 47 Staphylococcus aureus strains isolated from bloodstream in PLA General Hospital were collected from January 2006 to December 2008. Susceptibility of the strains to 11 antimicrobial agents was detected and DNA homology of them was analyzed with Rep-based DiversiLab(TM) Microbial Typing System. Panton-Valentine leukocidin (PVL) gene was determined by PCR. For methicillin-resistant Staphylococcus aureus (MRSA) strains, the genotypes of SCCmec were determined and ST239 clone was screened with multiplex PCR. Multilocus sequence typing (MLST) was used to determine the STs of the selected isolates. RESULTS: In the 47 Staphylococcus aureus isolated from blood, methicillin-resistant strains accounted for 51.1%, all belonged to SCCmec III type, with only 2 pvl gene positive strains identified. 12 different patterns (A-L) were found among 47 strains with Rep-PCR. All MRSA strains clustered in the A and B subtypes. CONCLUSION: Most MRSA strains isolated from blood in PLA General Hospital belonged to ST239-MRSA-SCCmec III clone. DiversiLab(TM) Microbial Typing System could provide a rapid and effective method to investigate the molecular epidemiological characteristics of Staphylococcus aureus in the hospital settings.

[Quantitative PCR for diagnosis of invasive fungal infections in patients with hematologic malignancies].

This study was aimed to establish the method of quantitative PCR (q-PCR) of fungi in peripheral blood for diagnosis of invasive fungal infections in patients with hematologic malignancies, and to preliminarily assess the diagnostic value of this method. The 18S rDNA-ITS1 area of high consensus sequence of fungi was selected to design primer and probe, the DNA of fungal species was extracted and q-PCR was performed to evaluate the sensitivity and specificity of the primer and probe. The standard product of fungal DNA was prepared by using pGEM-T plasmid and the fungal DNA in blood of patients was quantitatively detected. The results showed that the positive was found in 12 Aspergillus and 14 Candida species according to q-PCR detection, while there was no significant difference of fungal distribution between plasma, mononuclear cells and leukocytes (p<0.05). Receiver-operating characteristic analysis of q-PCR showed that the cut-off value for clinical diagnosis of invasive fungal infection was 8 copies/ml whole blood, its sensitivity, specificity, positive and negative predictive value and kappa were 0.84, 0.9, 0.955, 0.692 and 0.679 respectively. It is concluded that the fungal q-PCR assay may be used as an early diagnostic method for invasive fungal infections in patients with hematologic malignancies.

Susceptibility patterns and molecular epidemiology of multidrug-resistant Acinetobacter baumannii strains from three military hospitals in China.

To date, little has been reported on the susceptibility patterns and molecular characterisation of multidrug-resistant Acinetobacter baumannii (MDRAB) clinical isolates from different Chinese military hospitals. In this study, 49 MDRAB strains were collected from three military hospitals during 2007. The minimum inhibitory concentrations (MICs) of 13 antibiotics were determined for each strain. Genotyping and dendrogram analysis of MDRAB strains were performed using the repetitive sequence-based polymerase chain reaction (rep-PCR) DiversiLab Microbial Typing System. PCR screening was carried out to investigate the distribution of various genes contributing to each resistance phenotype in the main clonal types. The rates of resistance to the majority of antibiotics tested varied between 75.5% and 100%, with the exception of polymyxin B. Two DiversiLab rep-PCR clones (A and B) were widespread in three hospitals in different cities, one clone (D) existed only in two hospitals located in the same city (Beijing), and the other two clones (C and E) were present in only one hospital. In addition, this study shows a high distribution of intI1, ISAba1, bla(OXA-23), bla(ADC), adeB, adeJ, abeM and tet(B) genes, which mediate resistance to structurally unrelated antimicrobials in MDRAB isolates. These results suggest that all isolates were resistant to at least three classes of antibiotics. In addition, clonal dissemination among the three hospitals located in two different cities in China, previously documented in many regions of Europe and Asia-Pacific nations, emphasises the epidemic potential of these MDRAB isolates.

[Current approaches to diagnosis and treatment of invasive fungal infection in HSCT recipients].

Invasive fungal infections (IFI) are a kind of the most severe complications after hematopoietic stem cell transplantation (HSCT), Candida and Aspergillus are common causes. Because of immunosuppressive therapy, ablative conditioning regimen, acute or chronic graft-versus-host disease, long-term treatment of broad-spectrum antibiotics and cytomegalovirus infection, IFI has increased in the past few years. Invasive mould infection is a major cause of morbidity and mortality in HSCT recipients. Methods for early diagnosis of IFI include clinical and laboratory examinations, as well as characteristic radiography. Voriconazole is the first-line antifungal agent for prevention of IFI. Combination therapy of two antifungal compounds such as azoles or amphotericin B with echinocandins have shown a good effectiveness and may be a promising future strategy for antifungal treatment. In this review, the early diagnosis and treatment of IFI in HSCT recipients are summarized. As for early diagnosis of IFI, the laboratory diagnosis techniques such as GM test, G test and PCR techniques are discussed. As for prophylaxis and treatment of IFI, the prophylaxis treatment, empirical treatment, preemptive treatment, targeted treatment, combined treatment and immunologic treatment are discussed.

[Study on the molecular characteristics of multidrug-resistant Acinetobacter baumannii].

OBJECTIVE: To investigate antibiotic resistance, carbapenemase genotype and the molecular epidemiology of multidrug-resistant Acinetobacter baumannii (Aba) collected from 3 military hospitals in China. METHODS: The minimum inhibitory concentrations (MIC) were examined by ager dilution method. Genotypes of carbapenemases were amplified by multiplex PCR and its products were sequenced. PCR was used to detect per gene. Homology of the resistant isolates was analyzed by pulse-field gel electrophoresis (PFGE). RESULTS: Among the 64 MDRA strains, 78.1% (50) strains possessed bla(OXA-23) gene, 89.1% (57) carried Class 1 integrase gene, 39.1% (25) with bla(PER-1) gene, and 1 strain with bla(OXA-58-like) gene. PFGE showed that 13 (A, B, C, D, E genotype) different clones were identified in these strains. A, B, and U clones were the predominant clones in three hospitals, respectitively. CONCLUSION: Outbreaks of multidrug-resistant Aba occurred at 3 military hospitals with the most prevalent carbapenemase as OXA-23 enzyme. OXA-58 type of carbapenemase and per-1 in Aba were also isolated.

[Transmission and molecular characteristics of carbapenem-resistant Acinetobacter baumannii].

OBJECTIVE: To study the mode of transmission and molecular characteristics on carbapenem-resistant Acinetobacter baumannii strain. Strains were isolated from different parts of samples in various patients. METHODS: Clinical information of carbapenem-resistant Acinetobacter baumannii isolates were stored and analyzed by WHONET 5.4 software. The transmission and pathopoiesis of the strains were learned through case file review. Genotypes of isolates were identified by pulse-field gel electrophoresis (PFGE) and genes of carbapenemase were detected by multiple PCR, in order to find molecular characteristics and relatedness between strains. RESULTS: 29 stains of Acinetobacter baumannii resistant to carbapenem were isolated from 2 or more kinds of samples among 13 patients'. Two genotypes were identified by PFGE: genotype A was obtained from 22 isolates in 11 patients and genotype B was obtained from 7 isolates in 4 patients. PCR amplification showed that all strains possessed OXA-23 gene except 1, and all strains possessed Integrase gene I except 3. CONCLUSION: There were 2 different genotypes from 29 strains of carbapenem-resistant Acinetobacter baumannii with Genotype A as the main type. OXA-23 carbapenemase gene and integrase gene I were detected from most of the isolates. All the strains could be easily transmitted in the body of the patients and among patients, hence becoming the epidemic pathogen of iatrogenic infection.